Cyclin-dependent Kinase Inhibitors Limit Induced Pluripotent Stem Cells Generation

Pluripotent stem cells (PSCs) have emerged as new targets of researches due to their remarkable capability of giving rise to many types of cells in the body and a potential therapeutic method in regenerative medicine. PSCs can be categorized based on origins into two types:embryonic stem cells (ESCs) and adult stem cells (ASCs). As for pluripotency, when ASCs isolated from individual organs such as bone marrow (hematopoietic stem cells) or neural tissue (neural stem cells) can differentiate into cell types that belong to the systems where the ASCs dwell, ESCs derived from the inner body of embryo however can form all three layers of cells or even give rise to a living offspring. This ability is called pluripotency. Despite the pluripotent nature of ESCs that self-renew infinitely in culture while retaining the capacity of giving any type of cells in the body, their application in the clinic is still restrained mostly because of ethical issues of using ESCs by destroying human embryos and the fact that they can only be derived from early-stage of embryos, which precludes the establishment of autologous cell line from patients.In order to circumvent the hurdles that keep studies focusing on PSCs behavior rather than their clinical potential, methods of generating stem cells from somatic cells, instead of harvesting them directly from bodies, have been invented. Among those are nuclear-transferred embryonic stem cells (NT-ESCs), pluripotent hybrid cell and induced pluripotent stem cells (iPSCs). NT-ESCs can be generated by replacing the nuclear of oocyte with one derived from a somatic cell, after which some unknown factors in ooplasm act to reverse the epigenetic state of donor somatic cells back to that of a pluripotent stem cell. NT-ESCs render ESCs in the ways that they have similar patterns of methylation of DNA as well as comparable capabilities to self-renew and proliferate. However, high inefficiency owing to incomplete reprogramming and technical limitations prevent its first success with human cells. Pluripotent hybrid cell, as its name may imply, can be generated from fusing a somatic cells with an ESC, within which the epigenetic state of the fused cell can be reprogrammed back to a primitive stage as what is done in NT-ESC. Although generation of pluripontent hybrid cells doesn’t require genetic modification and less technically-challenging, the resultants cannot be used in human as they are tetraploid. The generation of iPSCs, on the other hand, doesn’t require any involvement of ESCs. Pluripotency can be re-established directly in somatic cells by forcing expression of the defined four transcription factors (Oct4, Sox2, Klf4 and c-Myc). After a transient expression of these factors delivered by viral vectors and subsequent epigenetic modification within somatic cells, endogenous genes governing the pluripotency as of ESCs such as Oct4 start to be expressed. In this way, pluripotency can be re-established in somatic cells. Given that any somatic cells, such as skin cells, can be reprogrammed, generating patient-specific iPSCs becomes possible.Despite a promising future of patient-specific iPSCs as a therapeutic method in regenerative medicine, obstacles such as low reprogramming efficiency and unknown molecular mechanisms underlying the reprogramming hinder iPSCs application on patients. One of them is the signal pathway of p53 and its downstream effectors, activation of which during the reprogramming induces apoptosis and cell cycle arrest. In order to better understand the mechanism behind the regulation on cell cycle of reprogramming cells and improve the reprogramming efficiency so as to refine the technology, our study explored changes of cell cycle status in the reprogramming cell and located two other regulative molecules involved in the regulation of cell cycle of reprogramming cells and revealed their roles in iPSCs generation.Part ⅠGeneration of induced pluripotent stem cells from somatic cellsBackground:Pluripotency can be re-established in somatic cells by forcing expression of the defined transcription factors (Oct4, Sox2, Klf4 and c-Myc) and the resultant are called induced pluripotent stem cells (iPSCs). Compared to other techniques of generating stem cells from somatic cells, this method is simple and robust, and can be performed on readily available skin cells to generate patient-specific iPSCs.Aim:to generate iPSCs from somatic cells and to test their pluripotencyMethods:iPSCs were generated from murine embryonic fibroblasts (MEFs) by transduction of the four transcription factors (Oct4, Sox2, Klf4 and c-Myc) delivered by retrovirus prepared from competent TOP 10 cells. Morphologic changes of reprogramming cells were recorded in the process. Newly formed iPSCs colonies were isolated and expanded individually until they became stable cell lines. The expression of ESCs markers (Oct4, Sox2, Nanog and SSEA-1) was measured as well as their ability to form terotomas. Chimera formation with newly formed iPSCs so as to test their contribution to normal development was also tested.Results:After 14 days of reprogramming, MEFs finally became iPSCs as they gained the morphology characteristics of ESCs and expressed ESCs markers (Oct4, Sox2, Nanog and SSEA-1). These cells can differentiate into all three layers of cells, evidenced by the formation of teratomas 3 weeks after injected subcutaneously into NOD/SCID mice. The substitute pregnant mice receiving embryos implanted with these iPSCs lines gave birth to offsprings containing skin cells that developed from implanted iPSCs, proving the ability of iPSCs to contribute to normal development.Conclusion:Somatic cells can be reprogrammed to pluripontent stem cells by retrovirus-delivered four transcription factors (Oct4, Sox2, Klf4 and c-Myc). The resultants iPSCs resembled ESCs in that they expressed ESC markers, can differentiate into three layers of cells, and were able to contribute to normal development when implanted into embryos.Part IICyclin-dependent kinase inhibitors limit induced pluripotent stem cells generationBackground:Despite a promising future of patient-specific iPSC in regenerative medicine, obstacles including low reprogramming efficiency and unknown molecular mechanisms underling the reprogramming prevent its application on patients. Among those, one is the signal pathway of p53, activation of which during the reprogramming limits iPSCs generation mainly by inducing apoptosis and cell cycle arrest. The latter is mediated by activation of p21, a member of the cyclin-dependent kinase inhibitors (CDKIs). It’s been well-studied that CDKIs including CIP/KIP and INK4 families are the main molecules involved in cell cycle regulation. As p18 and p27, member of INK4 family and CIP/KIP family, respectively, restrain cell cycle and limit self-renewal of hematopoietic stem cells, whether they also play a role in iPSCs generation remains to be addressed.Aim:The role of p18 and p27 in the iPSCs generationMethods:The cell cycle status of MEFs and ES cells as well as that of MEFs during reprogramming was measured by using SSEA-1 as a stem cell marker. mRNA expression of p18 and p27 among ESCs, iPSCs and MEFs as well as that in the reprogramming process were also measured. Reprogramming efficiencies of MEFs derived from p18 knock-out (KO), p21 KO, and p27 KO trans-genetic mice were compared. In addition, the reprogramming efficiencies of MEFs of p18 KO or p27 KO co-transduced with p18 or p27 over-expression vector, respectively, were compared. By using chemical compound Roscovitine or PD-0332991, the effect of direct inhibition of CDK2 or CDK4/6 on the reprogramming efficiency of p27 KO or p18 KO MEFs was measured respectively.Results:most ES cells were in S phase as compared to MEFs. In the reprogramming process, SSEA-1+ cells reprogrammed with the four transcription factors were mostly in S phase rather than G1, indicating that active cell cycling is required for cells undergoing reprogramming. In consistent with cell cycle profile, mRNA expression of p18 and p27 in the reprogramming process were constantly lower than controls as the cells were being reprogrammed. Also, their low expression pattern was seen in ES and iPSCs as compared to MEFs, confirming that a active cell cycle is one of characteristics of pluripotent stem cells. Absence of either p18 or p27 enhanced iPSCs generation to an extent comparable to that of p21 deficiency, evidenced by increased numbers of iPSC colonies formed from p18 or p27 KO MEFs. In addition, the enhanced effect of their absence on iPSC colony formation can be mitigated by forcing expression of p18 or p27 in p18 or p27 KO MEFs, respectively. Direct inhibition of either of Cyclin D-CDK4/6 or Cyclin E-CDK2 complexes by PD-0332991 or Roscovitine, respectively, reversed the effect of p18 or p27 deficiency on iPSC colony formation, indicating deficiency of either p18 or p27 facilitated iPSC generation by lessening their inhibition on Cyclin/CDK complex and thus allowing the cell cycle to proceed.Conclusion:As cell cycle inhibitors, the presence of p18 and p27 in somatic cells limits iPSCs generation by inhibiting on cyclin/CDK complex and thus restraining the cell cycle. Inhibition of p18 or p27 thus can enhance the reprogramming efficiency, indicating they can be potential targets in future studies.